Recovery and purification of sterols



- decomposed to liberate'the sterol.

Patented Jan. 2, 1951 RECOVERY 2,536,753 7 AND PURIFICATION OF STEROLS Hendrik Willem Knol, Veenendaal, Netherlands,

assignor to V. Veenendaalsche Sajet-en Vijfschachtfabriek Voorheen Wed. D. S. Van

- Schuppen & Zoon, Veenendaal, Netherlands No Drawing. Application September 27, 1948, Serial No. 51,489. In the Netherlands February This invention relates to recovery and purification of sterols, or more specifically to processes of separating sterols, such as cholesterol, from new materials containing free sterols and other unsaponifiable matter including other alcohols, wherein such'new materials are admixed with zinc chloride in amount sufficient to form additive compounds with the sterols and other alcohols present, and the resulting sterol-zinc chloride addition product is separated from the other substances present in the reaction mixture, the sterol-zinc chloride addition product being then My invention further comprises processes wherein the amount of zinc chloride used preferably amounts to from 2 to 8 moles per mole of sterol, i. e. from about four to sixteen times the amount of zinc chloride required by theory to react .with the amount of sterol present in the raw material and the reaction takesplace in the presence of a low boiling solvent, preferably a hydrocarbon solvent such. as benzene; all as more fully set forth and as claimed.

In the past, sterols have been conventionally separated from sterol-containing raw materials by the digitonine process. This process, however, is very expensive owing to the unavoidable losses of digitonine. Another method is described in the U. S. Patent No. 2,362,605 wherein oxalic acid is reacted with a sterol containing material E and the reaction product obtained is separated from the reaction mixture and then decomposed to liberate the sterol. More recently ithas been suggested to use various metalsalts as reagents for separating sterols from natural, or prelimi ing sterols from raw materials containing the same, by a process involving the formation of metal salt sterol addition products. These addition products are separated from the reaction mixture and the sterol is readily liberated therefrom by boiling in water.

The metal salt-sterol addition products formed in the Overhofl et a1. process can be represented by the formula MX-Sterol, :wherein MX represents a. molecular equivalent of a metal salt. In

8 Claims. (01. zed-397.2

the case of zinc chlorid the corresponding addition' product would have the formula ZnClz (SteroDz.

In theacknowledged Overhofi et al. application it is-recommended that the metal salts be added to the raw materials containing free sterols-in amount suflicient to react with the sterols. Best results were obtained with manganese chloride and rather inferior results with zinc chlorides.

yield of only 60.6 per cent of cholesterol having a purity of 61.2 per cent being obtained bythe treatment of wool grease or wool wax alcohols. In experiments with the Overhofi et a1. process I have discovered that in the case of most metal salts the use of the metal salt in excess of theoretical proportions results in the recovery of ;a sterol of inferior purity. I believe this to be due to the precipitation by these metal salts-of insoluble addition products of the metal salts with the extraneous alcohols present in the raw materials. In the treatment of wool wax alcohols, i. e. the unsaponiflable fraction of wool grease, for ex-'- ample, the metal salts cause the formation of insoluble addition products of the wool wax alcohols as well as of the cholesterol present. It is then very diflicult to separate the metal sa1tcholestero1 addition product from the addition products of the other alcohols. e I have made the surprising discovery, however, that zinc chloride is unique among the metal salts tested in that the use of an excess of zinc chloride over theoretical proportions not only increases the yieldpf sterol recovered in the process but produces a surprisin increase in the purity of the sterolreoovered. The minimum quantity of zinc chloride required to produce optimum results varies with the quantity of extraneous alcohols present in the raw material to be treated. I have foundthat this minimum quantit should be at least suificient to convert all of the alcohols present, i. e'. the sterol as well'as extraneous alcohols,

to the corresponding addition products. Fortu nately an additional quantity of zinc chloride over this minimum does no harm and in the case of all the raw materials which I have tested I have foundit advantageous to add at least 2 moles of zinc chloride per mole of sterol present, ifepja quantity-4 times the theoreticalasbased on they v sterol present. In thecase of wool wax alcohols containing about 30 to 40,per cent cholesterol optimum yields and purity are obtained when from about l to 16 times the theoretical quantity of zinc chloride is used. I therefore prefer to employ from 2 to 8 moles of zinc chloride to 3. mole of sterol in the raw material treated.

I attribute the surprising increase of yield and purity obtained by the use of an excess of zinc chloride to the fact that the addition compounds formed between the zinc chloride: and the extraneous alcohols present are soluble in organic solvents for the sterol while the excess of zinc chloride in solution causes the solubility of the relatively insoluble zinc chloride-sterol.addition. leis.

product to become reduced still further. also true that the extraneous alcohols still present after a theoretical quantity o'f' ain'c chloride has been added produce asolubilizin jefiect'on the zinc chloride-sterol addition product. So when sufiicient zinc chloride is'a'dded to convert these extraneous alcohols toi'theiraddition prod "amount of' zinc chloride need not-beregulated carefully, since large excesses'do'no harm. All that is required is that enough'be presentto comtime with the sterol and also to combine with any other materials present, 'such" as alcohols, which form additive compounds with zinc chloride; The yield of sterol isincreased to the-extent-'that the other materials'preserit; mostly wool "wax' alcohols, react'with the zinc chloride and "are" thus immobilized with regard to their solubilizing effect onthe' sterol addition compound. Among the further advantages flowing from thepresent invention there may be ment oned 'the fa'ctthat the*reaction-proceeds-at least slowly 'a't -room temperature so that/temperature control is-not critical. And the presence ofwate rin the -reacting mixture makes no" difference; hence no 'prel iminary dryingof thematerials'to'be treated isnecessary. In practicingthe process the sterol cont'aining material andjthezincchloride are siinply'adniixed together in the'presence' of an or.- ganic solvent for. the sterol, or; as" a. modification,

the solvent is added afterthe react on hasiinished. Any of the'common' organictsolvents which dissolve sterols are operative butl'find that the hydrocarbon solvents, particularly those of aromatic character. are especially useful. Among the suitable liquid solvents .there may be men:

tioned ethyl alcohol, low molecular ethers, such as dimethyl and diethyl ethers, etc, low molecular. nitroparaffins, such as nitro methane, nitro ethane, nitropropanes, etc., halogenated hydrocarbons,,such as trichloroethylene, dichloroethane, chloroform, etc., liquid hydrocarbons, such as gas! Ioline; petrole um ether, benzene, toluene, iso-oc- -tane, .etc., Inthese .solvents the zinc chloride- 'steroladditionproducts are insoluble while the addition products of the extraneous alcohols, normally-present in the ,oleaginous rawmaterials vcontaining sterols, are soluble. Separation of the zinc chloride-sterol.:addition products is therefore. very simple and may be accomplished abymfiltration'; by'centri-fuging the mixture or by 4 permitting the precipitate to settle followed by decantation of the liquid.

If desired it is possible to employ a mixed solvent. Some mixed solvents are capable of dissolving both the sterol addition product and the extraneous alcohol addition products. If the process is conducted with such a mixed solvent it is possible to produce precipitation of the sterol addition product-after the' 'reaction by distilling ofi one of the solvents. When amixture of isooctane and ethyl alcohol is used, for example, it ispossible to distill off the latter azeotropically.

Myprocess can be applied directly to any of the natural oleaginous raw materials which contain free sterols. In most of these materials, howeventhe sterolsfoccur as their esters with high molecular fatty acids. In this case the sterols must beset free before the zinc chloride procedures. ,A- treatment with an aqueous caustic alkali for example will decompose the esters and form soaps of the fatty acids. Since the latter aresoluble they can be readily separated from the insoluble sterols and other unsaponifiable matter which includes hydrocarbons, dihydric alcohols; etc: It is the unsaponifiable matter which is treated with zinc chloride in-accordance with the present invention. 'The presence 'of soaps or of fatty acids is detrimental. to the process; hence these should'beremoved 'as com pletely'as possible: 1

As mentioned above" the zinc chloridetreat ment can be conducted if d'esire'diin-the absence of extraneous solvents. In-this. case the reaction mixture is. advantageously heated. and s the :nonster'ol' matter present thentmelts', forming "a' "li quid phase, and servesasasolvent; Upon coolingthe reaction mixture-sets to a solid butitYis relatively easy to separate the zinc chloridesterol addition product fromithis'solid. This'can be accomplished by extracting. the impurities by means of one of. the. solvents'zmentioned above. or by 'a diiferential solvent-solventextraction using immiscible solvents or. by;any .other suitable method.

. It: is usually more: advantageous. to add: atsufii cient amount of an inert organic solvent? to dis:- solve' the fatty material to be treated before" the additionof. the zinc -chforide; Th'ezmixture' can beheated if desired. Temperaturesfromabout 41 toare advantageouslbutthe reaction will take place: readily at room temperatures. After the zinc: chloride treatment it is then merely necessary to filter'oricentrifuge the reaction mix ture to recover'the zinc-ch'loride-sterol addition product: i

The zinc chloride-sterol addition product-can be decomposed in several ways. It is merely nec essary to treat-it with water or'with an aqueous solution, usually accompanied'by heating, which will result in solution of the metal salt and liberation of theinsoluble sterol. It is also possible to distill the'sterol from the addition product under a vacuum. The sterolthus recoveredusually con tains a considerable amount of residual impurities from which it' cen'be freed by fraction'ally crystallizing the sterol from a suitable solvent, such as ethyl acetate, allyl chloride or ethyl alcohol. I

In order for my invention to be more fully un derstood I shall give tables showing the eifect of temperature conditions, 'theinfluence of the reactiontime, the results 'obtained with various solvents and the effect of varying-amounts of 1 zinc chloride. Where, in the following description I refer to the excess of zinc chloride as fourfold, sixfold etc. I mean that this is four or six times (up to sixteen fold; or sixteen times). the amount of zinc chloride necessary for combining with the sterol as required by theory, as explained above, i. e. a fourfold excess means 2 moles of Zinc chloride per mole of sterol etc.---

INFLUENCE OF TEMPERATURE 25 g. wool wax alcohol with a cholesterol content of 30.8% were stirred for 30 minutes with 125 cm} 'iso-octane and a sixfold excess ZnCl: (3 moles per mole of cholesterol) at difierent temperatures. In Table I the cholesterol content of the product obtained and the yield are shown.

Table I Temperature Cholesterol Yield 0. Percent Percent 50 84 71 so 83 19 70 91 89 so 93 so 9o 94 92 100 97 95 INFLUENCE OF TIME 25 g. of wool wax alcohols with a cholesterol content of- 30.8% were stirred with 125 cm. -isooctane and a sixfold excess ZnClz (3 moles per mole of cholesterol) on a steam bath for different periods. In Table II the cholesterol content of the product obtained and the yield are shown in percentages.

Table II Time Cholesterol Yield Minutes Percent Percent INFLUENCE OF SOLVENTS 25 g. wool wax alcohols with a cholesterol content of 30.8% were stirred with 6-12 fold excess ZNClz at 80-100 C. in difierent solvents. Table III gives the percentage of cholesterol in the product obtained and the yield in percent.

Table III Cholesjolvent teml Yield Percent Percent iso-octanc 92 89 petroleum ether 60-80, less than 1% aromatic 88 82 1% arnm aric 94 89 15% ar 92 84 etroleum ether 80-110, 6% aromatic 92. S5 enzene 95 8.6 toluene 85 71 Xylene 84 4 69 trichloroethylene 99- 77 r 54 41 85 52 87 67 INFLUENCE OF THE QUANTITY OF BENZENE 25' g. wool wax alcohols with a cholesterol, content of 30.8% were stirred for 60-minutes ona.

steam bath with an eight fold excess ZNClz (4 moles per mole of cholesterol) and different quantities of'benzene. Table IV gives the'percentage of cholesterol of the product obtained and the yield in percent, when using a quantity of benzene fold excess ZnClz (3 moles per mole of cholesterol) on a. steam bath the results were:

Table V Isa-octane:

Wool wax Cholesterol Yield alcohols Percem Percent 1 r 00; 85 g 2 92.5 87.5 -3 95 90 INFLUENCE OF QUANTITIES OF CHLORIDE 25 g. wool Wax alcohols with a cholesterol con- ZINC 40 tent of 30.8% were dissolved in 225 cm. of benzene and heated during 60 min. on a steam bath with varying quantities of zinc chloride.

Table VI Moles ZnCh/mole Cholesterol Yield cholesterol Percent Percent l 12.5 2 86 77 3 82. 80 4 86 5 92 86 6 95 85 7 S9 83 Table VII Moles ZnCh/mole Cholesterol Yield cholesterol Percent Percent Emample 1. 325 g. (3 moles/mole cholesterol) of anhydrous zinc chloride (ZnClz) are added to 1000 g. wool wax alcohols with a cholesterol con-'- tent of 30.8%.: This mixture is heated during half an hour in a'steam'bath with constant stir ring. After .cooling' the reaction mass is stirred into-590.0: em? isoeoctane and then treated :inra: eentrifuge'z. The insoluble 'additiont :product is washed-l by stirring .in12'000. cm. ism-"octane: and then againLtreating in awcentrifug'e: Biy decontposition of the addition product withiwat'er 289 gt of a product are obtained-containing 9a.": cholesterol corresponding with a yield of 89%.

Example 2.25.0 g. of wool wax alcohols containing 30.8% cholesterol and iii-13 g. anhydrous ZnClz (3 moles/mole cholesterol) are dissolved together in a mixture of 400 cm. benzene and cm. alcohol. The alcohol is then removed from the solution by azeotropic distillation. After cooling the reaction mixture was treated in the same manneras described in Example 1. A yield of 6.20 g. of a productis obtained (cholesterol content 96.4%) corresponding witliapercentage yield of 77.5%.

Example 3 .I0 g. wool wax"alcohols containing 30.7% cholesterol and 8.13 g. anhydrous ZnGlz (3' moles/mole cholesterol) are dissolved together in a mixture 'of 250 cm. iso-octaneand 20 cm. alcohol. Thealcohol is removed from the solution by azeotropic distillation. After cooling the reaction mixture'is treated as above described.

t'l'i1usual manner. "The unsaponifiable part is dissolved"in'1000cm; benzene; 600 g. anhydrous ZnClz are added and the mixtureis stirred for half an hour at 90 C. After cooling the reaction mixture is poured into a washtower filled with benzene. After four hours'the precipitate is removed from the bottom and decomposed with water. After distilling off the benzene a yield is obtainedof 320 g. cholesterol (purity 95%).

Example 6 To 100 g. ofthe unsaponifiable part of tall oil (sterol content equals 17.7%) g. anhydrous ZnClzand .1000 cm. benzene are added. The mixture is heated on a steam bath one hour under continuous stirring; After cooling the reaction mixture is treated in a centrifuge. On decomposition of the addition product with water 36.5% of a product with 94% sitosterol are obtained. Yield 91%.

Eramplc, ...250 g. fishoil (saponification number 173.3) are saponified by boilingfor two hours with a solution-of 100g. KOH (87%) in" 1500 cm. water. The soa solution is extracted with petroleum ether whereupon the sterols dissolve in the ether. After evaporation of the petroleum ether 22.9 g. unsaponifiable substance remains with a sterol content ofj.24.5%. llfhe unsaponifiable substance is dissolved in .100 c' m. iso-octane Cir and 6 g. anhydrous ZnClz (sixfold excess) are 'fifiggigof a product are:obtained containing-858% .steroL. corresponding with ayield-.of. ..85.'8:% .If

thisgprocess repeated but.- with'gtheadiiitionrdf yieldis 0.1%

ExampZaX-AOD'. kg; 'woollwax alcohols with cholesterol: content ofi30.0 areidissolvedin2000l liters is'o octane. 130 kg.. anhydrous izn'Clzz (3; moles/mole cholesterol) are. added and the r'mixe. ture' is" heated for half an hour'ina vesselheated: by steam underiicontinuous.stirring. A'fter 'coole ing the reaction mixtureistreated-in acentrifuge. The rest is washed by once stirring in and treating in a centrifuge with 800 liters 'iso-o'ctane. After decomposition-of the addition prodnot with water'the solution iswashed with water until it is chlorine free and the iso-octane' is distilled off. 1 16" kg. ofa productare obtained? containing 98 3 cholsterol 'corresponding 'to a yield of 95%.

Example 9.25.0 g. wool wax alcohols with a cholesterol content of' 30.8% are dissolved in 25 cm. iso-octane. 8.13 g. anhydrous 'ZnClz (3 moles/mole cholesterol) are added and the mixture is heated for half an hour on a steam bath under continuous stirring. After cooling; the reaction mixture is treated in a centrifuge. The

1 rest is washedby once stirring in-and treating in a centrifuge with '=cm. iso-octane. After decomposition of the addition product with water the solution isweshed-With-water until it is chlorine free. The iso-octane is distilled off 7.57 g. of a product are obtained containing 8'9;6%':'cholestercl'corresponding to a yiel'd 'of"88.0 Example 10. -25.0 g. woolwax alcohols with-a cholesterolcontent of 30.8% are dissolved-in 2'25 cm. 'benzene. 1626' g. anhydrous zinc chloride 1 ('6 moles/mole cholesterol) are added and the" mixture is heated for one hour at 85 C. under continuous stirring. After" cooling the mixture is treated in a centrifuge, the precipitate is washed by stirring with 50 cm. benzene and again treated in a centrifuge. After decomposition of the addition-product with water and benzene or gasoline (petrol) the solution is washed with water until it is chlorine free. The solvent is distilled off. 6.97 g. are obtained of a product containing 94.5% cholesterol corresponding to a yield of 85.5%. I

Example 11.25 g. wool wax alcohols with a cholesterol contentof 30.8% are dissolved in 225 cm. benzene; 10.84- g. anhydrous ZnClz (4 moles/mole cholesterol) are added and the mix-;. ture is heated .for one hour to 20 C. under continuous stirring. The mixture is then treated in a centrifugathe precipitate is washed by stir ring with 50 cm. benzene and again treated in a centrifuge. After decomposition of the addition product with water and benzene or gasoline" (petrol) thesolution is washed with water until it is chlorine free. The solvent is distilled off. 7.0 g. of a product are obtained containing 95.8% cholesterol, corresponding to a yield of 87.0%.. h Example 12.2-5 g. wool wax alcohols with a cholesterol content of 30.8% are dissolved in 12'5- cm. petroleum-ether -80 (1% aromatic) (1048 g. anhydrous'ZnClz (4" moles/mole cholesterol) are added and the mixture is heated forionehourf at CY-under continuousstirring; aftericoo'b. ing the mixture is treated in a centrifuge,- the: precipitate is washed by stirring with 50' cm? petroleum ether and treated'again in a centrifuge." After decomposition of the addition product with water and benzene or gasoline (petrol) the solution is washed with water untilitlis chlorine free; The solvent isdistilledwoff. 7.3 g; of aproduct are-obtained containing. 93.8% cholesterol:-

corresponding.ztcrayyield:of.89;0%;

It will be obvious that the processes of the present invention can be used in the isolation and recovery of sterols from many sources, for example, cholesterol from wool grease, brain and spine tissue and other materials containing free cholesterol, stigmasterol from soya beans, sitosterol from cereal grains, ergosterol from yeast and phytosterol from tall oil. And it will be apparent to those skilled in the art that many modifications fall within the purview of the present invention.

What I claim is:

l. The process of separating sterols from materials containing free sterols and other nonsaponifiable matter including other alcohols, which comprises admixing such a material in liquid phase with zinc chloride, the amount thereof being at least sufficient to form addition compounds with the sterol and with the other alcohols present and amountingto from about '2 to 8 moles per mole of sterol present, separating the sterol-zinc chloride addition compound from the reaction products and decomposing the sterol-zinc chloride compound to liberate the sterol.

2. The process as in claim 1 wherein the reaction is conducted in the presence of a liquid hydrocarbon solvent.

3. The process as in claim 1 wherein the zinc chloride is added to a solution of the sterol-conmaterials containing the same at least partly in' the form of their fatty esters, the process which comprises subjecting such a raw material to a hydrolyzing step, separating the hydrolytic products into a saponifiable fraction and an unsaponifiable fraction, heating the latter fraction in liquid phase with from about 2 to 8 moles of zinc chloride per mole of sterol present, thereby forming addition products with the alcohols present, separating the sterol-zinc chloride addition product from the other reaction products, decomposing the said sterol-zinc chloride addition product to form the free sterol and recovering the latter.

6. In the recovery of cholesterol from wool grease, the process which comprises subjecting wool grease to a hydrolyzing step, separating the hydrolytic products into a saponifiable fraction and an unsaponifiable fraction, mixing the latter fraction with an inert organic solvent for cholesterol, adding from about 2 to 8 moles of zinc chloride per mole of cholesterol present and HENDRIK WILLEM KNOL.

REFERENCES CITED The following references are of record in the file of this patent:

FOREIGN PATENTS Country Date France July 19, 1943 Number 

1. THE PROCESS OF SEPARATING STEROLS FROM MATERIALS CONTAINING FREE STEROLS AND OTHER NONSAPONIFIABLE MATTER INCLUDING OTHER ALCOHOLS, WHICH COMPRISES ADMIXING SUCH A MATERIAL IN LIQUID PHASE WITH ZINC CHLORIDE, THE AMOUNT THEREOF BEING AT LEAST SUFFICIENT TO FORM ADDITION COMPOUNDS WITH THE STEROL AND WITH THE OTHER ALCOHOLS PRESENT AND AMOUNTING TO FROM ABOUT 2 TO 8 MOLES PER MOLE OF STEROL PRESENT, SEPARATING THE STEROL-ZINC CHLORIDE ADDITION COMPOUND FROM THE REACTION PRODUCTS AND DECOMPOSING THE STEROL-ZINC CHLORIDE COMPOUND TO LIBERATE THE STEROL. 